Korean J Physiol Pharmacol.  2019 Sep;23(5):345-356. 10.4196/kjpp.2019.23.5.345.

Docosahexaenoic acid reduces adenosine triphosphate-induced calcium influx via inhibition of store-operated calcium channels and enhances baseline endothelial nitric oxide synthase phosphorylation in human endothelial cells

• Vu TT
• Dieterich
• Vu TT
• Deussen A

• KMID: 2455811
• DOI: http://doi.org/10.4196/kjpp.2019.23.5.345

Abstract

Docosahexaenoic acid (DHA), an omega-3-fatty acid, modulates multiple cellular functions. In this study, we addressed the effects of DHA on human umbilical vein endothelial cell calcium transient and endothelial nitric oxide synthase (eNOS) phosphorylation under control and adenosine triphosphate (ATP, 100 µM) stimulated conditions. Cells were treated for 48 h with DHA concentrations from 3 to 50 µM. Calcium transient was measured using the fluorescent dye Fura-2-AM and eNOS phosphorylation was addressed by western blot. DHA dose-dependently reduced the ATP stimulated Ca²âº-transient. This effect was preserved in the presence of BAPTA (10 and 20 µM) which chelated the intracellular calcium, but eliminated after withdrawal of extracellular calcium, application of 2-aminoethoxy-diphenylborane (75 µM) to inhibit store-operated calcium channel or thapsigargin (2 µM) to delete calcium store. In addition, DHA (12 µM) increased ser1177/thr495 phosphorylation of eNOS under baseline conditions but had no significant effect on this ratio under conditions of ATP stimulation. In conclusion, DHA dose-dependently inhibited the ATP-induced calcium transient, probably via store-operated calcium channels. Furthermore, DHA changed eNOS phosphorylation suggesting activation of the enzyme. Hence, DHA may shift the regulation of eNOS away from a Ca²âº activated mode to a preferentially controlled phosphorylation mode.

Keyword

Adenosine triphosphate; Calcium; Docosahexaenoic acid; Human umbilical vein endothelial cell; Store-operated calcium channels

MeSH Terms

Adenosine Triphosphate
Adenosine*
Blotting, Western
Calcium Channels*
Calcium*
Endothelial Cells*
Humans*
Nitric Oxide Synthase Type III*
Phosphorylation*
Thapsigargin
Umbilical Veins
Adenosine
Adenosine Triphosphate
Calcium
Calcium Channels
Nitric Oxide Synthase Type III
Thapsigargin

Figure

• 

  Fig. 1 Calcium increase of human umbilical vein endothelial cell (HUVEC) induced by adenosine triphosphate (ATP).(A) Typical fluorescence image. Typical fluorescence of Fura-2AM loading cell image with 40× magnification. (B) Maximum of ATP (100 µM) induced calcium transient in HUVEC treated with 0, 3, 12, 50 µmol/l docosahexaenoic acid (DHA) (n = 4). (C) Kinetic of calcium transient in cells stimulated with ATP (100 µM) without and with treatment with 12 µM DHA for 48 h (n = 8). Cells were loaded with 5 µM Fura-2-AM. ANOVA; ***p < 0.0001 between DHA treatment and control.

• 

  Fig. 2 Effect of docosahexaenoic acid (DHA) on adenosine triphosphate (ATP)-induced endothelial nitric oxide synthase (eNOS) phosphorylation in human umbilical vein endothelial cell (HUVEC).HUVEC were treated with 12 µM DHA for 48 h. 100 µM ATP was used to stimulate eNOS for 1 min. (A) Total eNOS expression (n = 7). (B) Representative blot. (C) Phosphorylated eNOS at ser1177 residue (n = 5). (D) Phosphorylated eNOS at thr495 residue (n = 5). (E) Ratio of phosphorylated eNOS at ser1177 and phosphorylated eNOS at thr495 (n = 5). (F) Representative Western blot. The results show mean ± standard error of densitometric quantification of blots. ANOVA was used to test for differences. *p < 0.05, ***p < 0.001 vs. untreated group (no DHA, no ATP); #p < 0.05, §p < 0.05. NS, no significant difference.

• 

  Fig. 3 Effects of BAPTA and withdrawal of extracellular calcium on adenosine triphosphate (ATP) induced calcium transient.(A, B) Presence of BAPTA (10 µM, 20 µM) does not eliminate the effect of docosahexaenoic acid (DHA) on ATP-induced calcium transient (n = 3). (C, D) Elimination of extracellular calcium reduces the ATP-induced calcium transient and abolishes the effect of DHA on the calcium transient (n = 4). DHA 12 µM, ATP 100 µM; ANOVA/Tukey's post-hoc test was used: **p < 0.001; ***p < 0.0001, #p < 0.0001 between groups as indicated by brackets,. NS, no significant difference; CS, calcium-saturated; CF, calcium-free. For further information see methods.

• 

  Fig. 4 Effects of 2-aminoethoxydiphenyl borate (2-APB) on adenosine triphosphate (ATP) induced calcium transient and endothelial nitric oxide synthase (eNOS) phosphorylation of human umbilical vein endothelial cell.Cells were left untreated or treated with 12 µM of docosahexaenoic acid (DHA) for 48 h. (A, B) Effect of 2-APB (75 µM, 30 min at 37℃) on ATP-induced (100 µM) calcium increase. ANOVA/Tukey's post-hoc test, n = 4, ***p < 0.0001 vs. untreated group. (C–F) Effects on eNOS phosphorylation. Results show mean ± standard error of densitometric quantification of blots from 4 independent experiments. ANOVA was used to test for differences between groups. *p < 0.05, **p < 0.01 vs. untreated group; #p < 0.05, §p < 0.05. NS, no significant difference.

• 

  Fig. 5 Effects of calcium store-depletion by thapsigargin on adenosine triphosphate (ATP)-induced maximum calcium increase and endothelial nitric oxide synthase (eNOS) phosphorylation.(A, B) Maximum of calcium increase in presence of thapsigargin (2 µM) on the left and following ATP (100 µM) in presence of thapsigargin on the right. The calcium signal was recorded following thapsigargin (TG) for 450 sec before ATP was applied. Cells were either treated with docosahexaenoic acid (DHA) 12 µM for 48 h or left untreated (control), n = 3, 37℃. (C–F) Effects of TG on eNOS phosphorylation. Cells were stimulated with TG (1 µM, 10 min) before stimulation with ATP (100 µM, 1 min). Samples were harvested and stored at −80℃ for western blot. Results show mean ± standard error, n = 4. ANOVA/Tukey's post-hoc test was used to test for differences between groups. *p < 0.05, **p < 0.01, ***p < 0.001 vs. untreated group; #p < 0.05, §p < 0.05. NS, no significant difference.

• 

  Fig. 6 Effects of PPADS on adenosine triphosphate (ATP)-induced maximum calcium signal and endothelial nitric oxide synthase (eNOS) phosphorylation.(A, B) Maximum calcium signal in human umbilical vein endothelial cell treated with 12 µM docosahexaenoic acid (DHA) for 48 h or left untreated (control), n = 3, PPADS 100 µM for 30 min at 37℃. (C–F) Effects of PPADS on eNOS phosphorylation. Results show mean ± standard error, n = 3. Data was tested using ANOVA/Tukey's post-hoc test to compare different groups. *p < 0.05, **p < 0.01, ***p < 0.0001 vs. untreated group; §p < 0.0001 between DHA treated groups with and without PPADS; #p < 0.01 between PPADS treated groups with and without DHA. NS, no significant difference.

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